HU220784B1 - Extracts of shark cartilage having an anti-angiogenic activity and an effect on tumor regression, process of making thereof - Google Patents

Abstract

The present invention relates to shark cartilage extracts and to a method of producing the same, these extracts having anti-angiogenic properties (reduction of the area of blood vessels observed in vivo on experimentally induced tumors), tumor regressive activity in vivo as well as demonstrating a direct inhibitory effect on tumor cell lines. This process does not involve any denaturing solvent or product and does not involve the use of any enzymes. It consists of obtaining a blend of whole cartilage in an aqueous solution of neutral pH, preferably pure water, this blend being centrifuged and the pellet and supernatant kept for further processing. The pellet is lyophylized and tested for anti-tumor and anti-angiogenic activities in vivo and in vitro, with or without supernatant. The supernatant has been shown to have anti-angiogenic and tumor regressive activities in vivo. The composition of the supernatant has then been investigated by different ways. Fractionation of this supernatant led to the characterization of some of its active components. The fractions were tested for their direct in vitro activity on cancer cell lines. Therefore, it is assumed that the non-fractionated supernatant has such an in vitro activity. Lyophilization substantially destroys the activity of these liquid fractions while no such abolition is observed in the solid extract.

Description

FIELD OF THE INVENTION The present invention relates to antiangiogenic and antitumor shark porcine extracts and their preparation.

The cartilage is not a vascular tissue, so the question is whether they contain factors that inhibit vascularization. The cartilage tissue is also relatively resistant to tumor growth. The cartilage tumor, cartilage sarcoma, is the least vascularized solid tumor. Vascular formation is an important factor in tumor development. Distinct solid tumor nodules appear when tumor cells are able to displace the size of the nearby vasculature to meet their nutritional needs. Because of their role in tumor development, vascular stimulation factors, vascular inhibitory factors, and vasodilator agents that control tumor growth or have tumor regression have been investigated.

Langer et al., In 1976, discovered that the calyx cartilage cartilage contains a substance that inhibits vascularization in solid tumors (1976, Science, 193, 70-72). As it was successfully used as an antitumor agent, a greater amount of cartilage sources was sought.

Sharks are potential sources of antifungal agents, as their entire skeleton is made up of cartilage (6% of their body weight is cartilage compared to 0.6% of calves). A remarkable property of sharks is that they are less prone to tumor formation. Many theories have been developed to explain why sharks are so unlikely to develop tumors. Among other things, it has been shown that IgM antibodies are capable of immediately attacking any hostile material, and that sharks have macrophages that can distinguish normal cells from tumor cells and destroy the latter. Rosen and Woodhead have speculated that animals such as sharks and scabies are rarely tumorous because their tissues have an ionic strength as high as high body temperature [High shoot strength: Its Significance in Tumor Cells in Sharks and Rays] ) ”Medical Hypotheses. 6: 441-446 (1980)]. According to the authors, under these conditions, the immune system is close to 100% immune. Sharks also produce aminosterol with antibacterial and anti-protozoal properties. Finally, Lee and Langer (Shark Cartilage Contains Inhibitors of Tumor Angiogenesis, 1983, Science 221, 1185-1187) and Folkman and Klagsbrun have shown that sharks produce reuptake inhibitors (Angiogenic Factors, Science 235, 442- 447 (1987)]. Lee and Langer isolated this material under denaturing conditions by extraction from shark cartilage (guanidine extraction), but this process can result in very long extracts (41 days) containing denatured elements. The above researchers found the active ingredient isolated from calves to have a molecular weight of about 16 kilodaltons (kDa), but did not provide an exact molecular weight value for the material recovered from sharks. This material has only been described as having a molecular weight of more than 3,500 Daltons (Da). Oikawa et al., ["The Increase Angiogenic Inhibitory Derived from Japanese Chark Cartilage (I). Extraction and Estimation of Inhibitory Activities Toward Tumor and Embryonic Angiogenesis, 1990, Cancer Letters 51, 181-186], using the same extraction procedure as Lee and Langer, but with a much shorter time (from 2 days to 41 days). According to Oikawa et al., The molecular weight of the material isolated from the shark cartilage ranges from 1000 to 10,000 Da. Schinitsky (U.S. Pat. No. 4,473,551) investigated an aqueous extract of coarse-powdered shark cartilage and found its fraction having a molecular weight greater than 100,000 Da, alone or in combination with glucoseamine, to be anti-inflammatory. The patent does not disclose any anticoagulant or antitumor activity for any of the components of the extract. Kuetner et al. (U.S. Patent No. 4,746,729) isolated a polymorphonuclear neutrophil (PMN) elastase inhibitor from bovine cartilage. This inhibitor was recovered from a fraction of the pores extract having a molecular weight greater than 50,000 Da. Fractionation was performed on Sephacryl S-200-οη, and fractions of 10-40 kDa with antielastase activity were collected from several fractions obtained. The active component has an isoelectric point of 9.5 and a molecular weight of about 15,000 Da. U.S. Patent No. 4,042,457 to Kuetner et al. Has shown that the bovine cartilage has a molecular weight of less than 50,000 Da but has no effect on proliferation. endothelial cells. Spilburg et al. (U.S. Patent No. 4,243,582) isolated two types of 65 kDa glycoprotein (e.g. 3.8) from bovine cartilage (guanidine extraction) which were found to have antitrypsin activity and inhibit endothelial cell growth.

Various biological effects of calf and shark cartilage are known, including effects on lysosomes, cell growth promoters, inhibitors of collagenase I and type IV, elastase and proteases such as trypsin, chymotrypsin and plasmin.

Methods for preparing shark extracts and fractions are known. Some of them produced coarse powdered cartilage without extraction (U.S. Patent 5,075,112), others using denaturing agents such as guanidine (U.S. Patent No. 4,243,582) or removing the surrounding cartilage by enzymatic reaction. muscles, nerves and blood vessels, and organic solvents have been used to remove fat (U.S. Patent Nos. 3,478,146, 4,350,682, and 4,656,137), thereby obtaining a pore extract. Others have simply prepared aqueous extracts from cartilage by removing the insoluble material (water in U.S. Patent No. 4,473,551; saline in U.S. Patent No. 4,746,729). In the latter case, the fractions of the given molecular weight were purified and studied (see above).

HU 220 784 Bl

In summary, shark cartilage is known to contain the vasoconstrictor component (s) and has typically been assayed by rabbit corneal pouch determination or chorioallantoic membrane (CAM) determination. The antitumor activity of fully powdered cartilage was tested directly on the tumor in vivo using a huran melanoma implant implanted in a nude mouse (U.S. Patent 5,075,112) and CAM. However, no evidence was found that cartilage extract directly affects tumor cells.

Artery formation does not only occur in cancerous growth. Numerous diseases or conditions affecting different physiological systems are associated with vascularization (the physical system involved is given in brackets after the disease), such as arthritis and atherosclerotic plaque (bone and ligaments), retinopathy, diabetica, neovascular glaucoma, trachoma and sarcoma. reabsorption (eye), psoriasis, scleroderma, hemangioma and hypertrophic scarring (skin), vascular adhesion and angiofibroma (blood system). Any new anti-angiogenic factor can be used in the treatment of these diseases, as in cancer therapy.

SUMMARY OF THE INVENTION The present invention relates to a process for the preparation of extracts having anti-vascular properties (reduction of the size of blood vessels in experimentally generated tumors) which inhibit tumor growth in vivo and directly inhibit the growth of cancer cell lines. These extracts can be obtained from cartilage (Squalus acanthias), commonly known as common spiny shark and black spiny shark. The process does not involve the use of a denaturing solvent or product or the use of an enzyme. In the process, a mixture of whole cartilage was prepared in an aqueous solution of neutral pH, preferably pure water, centrifuged, and the resulting pellet and supernatant set aside for further processing. The pellet was lyophilized and tested for in vivo and in vitro anti-tumor and anti-vascular activity, with or without the supernatant. The supernatant showed angioprotective and antitumor activity in in vivo studies. The composition of the supernatant was examined in different ways. The supernatant was fractionated and its active components were characterized. The direct in vitro effect of the fractions on cancer cell lines was investigated. It is expected that the unfractionated supernatant has similar activity in vitro. Lyophilization destroyed the activity of these liquid fractions. It is emphasized that a clear distinction should be made between solid and liquid extracts and their fractions.

The invention relates to the cartilage lyophilisate, the solid extract, the liquid cartilage extract and the liquid fractions thereof and to the preparation thereof.

Finally, the invention relates to the use of cartilage extracts in the treatment of vascular dependent diseases. Preliminary clinical trials have shown that pharmaceutical formulations containing concentrated, non-fractionated liquid extract improve the condition of patients with vascular dependent disease. Studies on dermatological disorders such as psoriasis and a case of prostate cancer have been successful. Acne is a dermatological disorder that is not classified as a vascular disease, but has been treated with surprising success. Excessive vascularization is a known feature of scarring of burns. To prevent this phenomenon, preparations containing cartilage extracts are currently being investigated. The invention also relates to pharmaceutical compositions comprising a liquid cartilage extract.

Some embodiments of the present invention will be described in more detail below and illustrated by the following figures, which are intended to illustrate the invention, but are not limited thereto.

Figure 1 shows the inhibitory effect of increasing shark cartilage dose (solid extract) on ZR75-1 and MCF-7 cells;

Figure 2 shows a dose response curve of the amount of MCF-7 cells in DNA in the presence of increasing concentrations of estradiol with and without two different cartilage lyophilisate concentrations;

3A. 3B and 3B. Figures 3A and 4B show a comparison of liver sections of advanced breast cancer rats fed artificially with a combination of cartilage lyophilisate and supernatant (Figure 3B) or to which only water was added (Figure 3A) (200x magnification);

4A. 4B and 4B. Figures 4 to 5 are comparisons of kidney sections of advanced mammary gland cancer rats fed artificially with a combination of cartilage lyophilisate and supernatant (Figure 4B) or water only (Figure 4A) with no detectable poisoning (200x magnification);

5A. 5B and 5B. Figures 5A and 5B show a comparison of lung sections of advanced mammary gland cancer rats artificially fed with a combination of cartilage lyophilisate and supernatant (Figure 5B) or water only (Figure 5A), with no cells peeled in the wells (200x magnification);

6A. 6B and 6B. Figures 6A and 5B show a comparison of cancer cells in advanced mammary gland cancer rats fed artificially with a combination of cartilage lyate and supernatant (Fig. 6B) or water-only (Fig. 6A), showing the effect of decreasing blood vessel size and size. fabric structure is denser (120x magnification);

FIG. 6B and 6B. Fig. 3 is a histogram derived from cartilage extract3

EN 220 784 B1 discloses the effect of Bl on tumor blood vessels;

8A. and 8B. Fig. 4A shows the electrophoresis profile of liquid fractions separated by rotorph under non-denaturing conditions. Molecular weight markers appear on the left, along with a crude permeate sample prior to fractionation for comparison with isolated fractions;

9A, 9B, 9C. and 9D. Figures 9A and 9C show a significant improvement in the condition of two patients with psoriasis treated topically with a topical formulation containing an effective amount of liquid cartilage extract (Figures 9B and 9D) compared to their initial condition (Figures 9A and 9C); 9B and 9B. 9C is a hyperkeratosis and FIG. and 9D. Fig. 4A to a non-hyperkeratosis patient.

Healthy black spiny sharks and common spiny sharks were harvested after capture. Their muscle and connective tissues were removed with ethanol-purified scalpels and scissors. The cartilage was then frozen at -20 ° C in plastic bags for later use in vacuum packaging.

The cartilage was thawed at 4 ° C. Grinded three times with an equal volume of purified water by inverse osmosis and 0.1 µm filtration on an ethanol-purified meat grinder to give the first mixture (w / v). Instead of water, various neutral aqueous solutions may be used which do not undergo lysis or denaturation of the cartilage component.

The mixture was then homogenized with an industrial mixer at a maximum speed of 4 ° C for ten minutes. The homogenate was liquefied by treatment with a Polytron disintegrator at 4 ° C for ten minutes. In this step, a particle size of less than 500 µm was obtained. The mixture was centrifuged at 13,600 x g for 15 minutes at 4 ° C. The resulting pellet was lyophilized for 24-48 hours. This gave the first fraction, hereinafter referred to as lyophilisate or extract.

The supernatant was filtered through a 24 µm Whatman filter to remove particles interfering with ultrafiltration column filtration. The filtered material was then ultrafiltered on a 500,000 Da tangential flow filter column at 4 ° C. The supernatant was filtered through a 0.22 µl sterile filter and filled into sterile vials for later use. This fraction is hereinafter referred to as the supernatant or liquid extract.

Alternatively, a more efficient method of recovering the lyophilisate and the supernatant was developed. The centrifugation step at 13,600 xg for 15 minutes and subsequent filtration on a Whatman filter were replaced by centrifugation in a CEPA centrifuge equipped with a 30 pM porosity nylon bag at 3,000-4,000 χ g. In this way, 20 kg / 201 of the composition can be centrifuged for 30 minutes, resulting in 21 liters of supernatant. The resulting aqueous volume is even greater than the initial volume of water, which means that it also includes part of the cartilage water content. The lyophilisate and the supernatant may have the following composition:

lyophilisate: greases 7.35% ¹ proteins 46.2% ² humidity 20.4% Sodium 4.16 mg / g ³ Potassium 2.64 mg / g Calcium 114 mg / g Magnesium 1.49 mg / g Zinc and iron traces Supernatant: greases 0.10% ¹ proteins 8 mg / ml ² humidity 98.8% Sodium 33.6 mg / 100 g ^: Potassium 39.2 mg / 100 g Calcium 2.0 mg / 100 g Magnesium 1.1 mg / 100 g Zinc and iron traces

' ² Measurements are published in AOAC Official (1984)

16,219-220 and 2,055.

³ Measurements were performed according to the SAA procedure.

The protein content was determined by the Kjeldahl method, which is actually based on the measurement of organic nitrogen (N). The equivalent protein content in organic nitrogen can be determined from the following equation:

Protein content (mg / ml) =% Nx 6.25 / 100.

Carbohydrates are undetectable but are believed to exist in the form of proteoglycans and / or mucopolysaccharides in each extract. It is possible that these compounds are included in the measured moisture content, since the moisture content of the lyophilisate, determined on the basis of hydroxy groups, is unexpectedly high. The measured moisture content of 20% is approximately the same as the percentage carbohydrate retrieved from the cartilage, whereas the moisture content of the lyophilisate should be close to 0%, but this assumption is not yet proven.

Sterility was assured using USP XXI1:

1) Laboratoire de Généé Sanitaire du Quebec Inc. 1090, l'Escarbot, Center Industriel St-Malo, Quebec GIN 4J4; and

2) Northview Laboratories Inc. 1880, Holste Road, Northbrook IL, 60062 U.S.A. FDA registration no. 14-18028.

Effects on LIOFILISATE In vitro study

Assays were performed on hormone-dependent MCF-7 and ZR75-1 cancer cell lines [ATCC (R)

The date of their filing was 22-HTB April 5, 1982 and 1500-CRL (June 19, 1979), respectively.

ZR75-1 cells

BASAL RPMI medium containing no phenol red RPMI 1640 (Sigma R8755), 17.875 g Hepes (free acid; Sigma H0763), 0.55 g sodium pyruvate (Sigma P5280) and 10 g NaHCO _{3 in} 5 L pure water, and the pH of the solution was adjusted to 7.40 with NaOH.

Unused solution was stored protected from light because it contains photosensitive substances.

The solution was filtered, filled into 500 ml sterile vials and stored at 4 ° C for up to three months.

Cell culture medium

Basal RPMI medium was supplemented with 10% v / v FBS (bovine fetal serum), 100 U penicillin G / 50 pg streptomycin sulfate (Sigma P0906) / ml medium, 2 mM L-glutamine (Sigma G1517) and 1 nM E ₂ (p-estradiol Sigma E8875).

Experimental medium

Basal RPMI medium was supplemented with 5% FBSA (charcoal adsorbed bovine serum), 2 mM L-glutamine, 100 U penicillin G / 50 pg streptomycin sulfate / ml medium and 50 ng / ml insulin (Sigma). To this medium was added to the above-described lyophilizate and increasing concentrations of various concentrations of estradiol (10 @ ^-5 M ^1210).

MCF-7 cells

BASAL DME-F12 medium

DME-12 medium (containing no bicarbonate and phenol red; Sigma) was prepared according to the manufacturer's instructions in pure water. To one liter, 1.2 g of sodium bicarbonate was added and the pH was adjusted to 7.40 with NaOH / HCl. The solution was filtered, filled into 500 ml sterile vials and stored at 4 ° C for up to three months.

Culture media

Basal DME-F12 medium was supplemented with 10% (v / v) FBS (bovine fetal serum), 100 U penicillin G / 50 pg streptomycin sulfate / ml medium, 2 mM L-glutamine (Sigma) and 1 nM E ₂ (estradiol).

Experimental medium

Basal DME-F12 medium was supplemented with 5% FBSA (dextran-charcoal absorbed fetal bovine serum), 2 mM L-glutamine, 100 U penicillin G / 50 pg streptomycin sulfate / ml medium and 50 ng / ml insulin ( Sigma). Lyophilisate and estradiol were used at the concentrations indicated for ZR75-1 cells.

Production of FBSA

Fetal bovine serum was mixed with 1% (w / v) charcoal (decolorizing carbon).

To the charcoal serum solution was added T70-dextran solution to a concentration of 0.1% (w / v). The mixture was stirred overnight at 4 ° C. After centrifugation at 10,000 g for 30 minutes at 4 ° C, the serum was decanted and repeatedly mixed with charcoal and dextran. It was then stirred at room temperature for three hours and centrifuged again. Serum was inactivated by heating at 56 ° C for 20 minutes, then sterile filtered and added to a sterile conical Falkon tube.

ZR75-1 and MCF-7 cells were grown on 24-well plates to 20,000 cells / well or on 6-wells to 150,000 cells / well and treated with various amounts of lyophilizate prepared as described above. Each experiment was repeated three times. The cell culture medium was replaced with fresh medium every other day. The cells were cultured in a constant humidity, 5% CO ₂ atmosphere in a 37 ° C incubator for the first, second, third or fourth experiment, respectively, for 17, 7, 3 or 3 days. Inhibition of cell proliferation was determined by direct counting of cells or by measuring the total DNA content of the well.

Liofílizátumkoncentráció Cellular inhibition (%) MCF-7 ZR-75-1 Experiment 1: 17 days 1 mg / ml 1.5 2.00 5 mg / ml 14.33 33.6 10 mg / ml 62.66 90.8 Experiment 2: 7 days 1 mg / ml 3.73 0.97 5 mg / ml 15.7 29.00 10 mg / ml 68.37 66.00 Experiment 3: 3 days 50 mg / ml 95.8 95.00 100 mg / ml 94.6 98.00 Experiment 4: 3 days 10 mg / ml 34.4 51.5 20 mg / ml 62.5 70.5 50 mg / ml 95.8 95 100 mg / ml 94.6 98

The above percentages of inhibition of cell proliferation show that the lyophilisate inhibits the proliferation of cells of both cell lines in a dose dependent manner.

Figure 1 clearly shows that the lyophilisate at 50 and 100 mg / ml clearly inhibited cell line proliferation after three days of treatment.

Figure 2 shows that in the presence of 10 ^-12 -10 ⁹ M estradiol, the treated cells were

They behaved similarly, ie they were not affected by these hormone rates. In contrast, above 1 nM, control cells reacted strongly and reached a value of 3.75 gg of DNA concentration of 10 ^-7 M estradiol in the presence (versus the control value without estradiol 0.69 gg). In cells treated with 30 and 50 mg / ml lyophilisate, the maximum DNA value was 1.9 and 1.8 gg, respectively. Figure 2 shows that the treated cells have an affinity constant (Km) for estradiol 3-16 times higher than that of the control cells at 30 and 50 mg / ml. This means that in the presence of lyophilized solid cartilage extract, a higher concentration of estradiol is required to achieve the same cell proliferation. Consequently, the extract reduces the maximal response (90% inhibition) of the treated cells to estradiol and increases their affinity constant.

Carrier tests

Four hundred 40-day-old female Sprague-Dawley rats (Charles River Co. St-Constant, Quebec) were acclimatized for 12 days. Then, 20 mg of DMBA (9,10-Dimethyl-1,2-benzanthracene, Sigma Chemical Co.) was obtained in 1 ml of cereal oil by artificial feeding. After three months, 240 rats with breast cancer were selected and divided into two groups. The first group was divided into five subgroups. Rats in the treated groups were treated with increasing amounts of lyophilisate extract in 3 ml of water daily for eight weeks, while the control group received the same amount of water. The second group was divided into four subgroups. Rats in the treated groups were also given daily lyophilisate (approximately 25 mg protein content) with or without supernatant in 3 ml of water for 10 weeks, and the control group received the same amount of water. One subgroup of rats in the second group was treated with lyophilisate at a concentration of 3000 mg / kg / day, and 3 ml of supernatant containing a lower dose of the supernatant (about 8 mg of protein in 1 ml of water) was injected intraperitoneally (ip).

The rats had a body weight of 151-175 g at the start of the two-week experiment, were fed ad libitum and drank water. Rats of the first group had an average tumor size of 0.9 cm and rats of the second group had a diameter of 0.6 cm.

Summary of results

Daily dose of cartilage extract with artificial nutrition % Inhibition of Nuclear Growth (reduction in tumor diameter compared to control) Experiment 1: Duration of 8 weeks 500 mg / kg / day 2% 1000 mg / kg / day 4% 3000 mg / kg / day 14% 5000 mg / kg / day 15%

Daily dose of cartilage extract with artificial nutrition % Inhibition of Nuclear Growth (reduction in tumor diameter compared to control) Experiment 2: Duration of 10 weeks 3000 mg / kg / day 12% 3000 mg / Kg / day + 3 ml of supernatant 18% 3000 mg / Kg / day + 3 ml supernatant + 1 ml inj., Ip. supernatant 20%

The results show that the lyophilisate contains an active component that is absorbed in the gastrointestinal tract and affects the size of the tumor. This effect may be an effect directly on the tumor cells or an action through inhibition of vascularization.

The results show that the supernatant reduces the tumor size by about an additional 5%, even in its highly diluted form (about 25 mg protein is present in 3 ml of the supernatant).

Histopathological examinations

To demonstrate that the cartilage extract's active molecules are non-toxic, animals used in the above in vivo experiment were decapitated and analyzed on the following tissues: liver, lung, kidney, heart, brain, muscle, and mammary gland. The listed tissues were fixed in Bouin's liquid for two days and then their fat content was removed. After dehydration with ethanol, fixed tissues were embedded in paraffin. Sections of these were placed on glass slides, stained with hematoxylin and examined under a microscope.

Histological examination did not show any adverse effect at the highest dose of lyophilisate (data not shown), nor in the supernatant combined assay (see Figures 3A, 3B, 4A, 4B, 5A and 5B).

Studies have shown that the lyophilisate and the supernatant are independent of tumor size reduction.

In cancerous mammary glands (Figures 6A and 6B), a significant reduction in the area of blood vessels is observed. The anti-vascular effect of the active molecules is confirmed by the results shown in the following photo and histogram:

Figure 7 shows that when the lyophilisate (po) supernatant (po + ip) combination is used (Figures 6A and 6B), the area of the blood vessels is reduced by 55%.

The decrease in tumor size is due to a significant decrease in vascularity, a direct effect on tumor cells, or a combination of the two. The antitumor activity of the extracts is described in detail above. Direct hypoplasmic activity on hormone dependent cells in vitro ta6

HU 220 784 BI, with in vivo confirmation still to be determined.

Since the above-described results showed that the supernatant enhances the effect of the lyophilisate on ZR75-1 cells, its components were further investigated.

Preparation of liquid fractions containing active molecules

The shark cartilage was collected and treated as described above, except that the concentration step was omitted. After centrifugation, the pellet was discarded and the supernatant was treated in the same manner as described above, until sterile filtration through a 0.22 µm filter.

The supernatant is hereinafter referred to as the crude filtrate, i.e. the product of ultrafiltration.

The crude filtrate thus obtained was separated by FPLC (Rapid Protein Liquid Chromatography).

FPLC conditions:

Column: Hiload 26 mm x 60 cm Sephacryl S-300

Pharmacia FPLC system

Each sample was filtered through a 0.22 µm filter before being applied to the column. Phosphate salt buffer (PBS) was used as the elution buffer, which was filtered and degassed for 15 minutes. The volume of sample applied to the column was generally 3.2 ml (maximum 13 ml) and the flow rate was 1 ml / min. Fractions of 10 ml were collected. The eluted compounds were detected by UV absorbance (280 nm). The calibration chart was prepared using a Sigma MW-GF-1000 calibration kit, the volume of the calibration sample being identical to the volume of the analyzed samples (3.2 ml). The sample elution volume was determined by plotting the molecular weights of the compounds in the calibration kit versus the elution volume minus the empty column volume. Empty volume was determined by injection of dextran blue (MT = 2,000,000).

The effect of the fractions was tested on ZR75-1 cells. Useful fractions were identified and their properties were confirmed by further assays (see below).

Supplemental characterization of the active components of the filtrate was performed on a rotophor (Biorad 170-2950; see below for isoelectrofocusing) and Amicon filters of various size ranges to separate the molecular weight fractions of 10-30 kDa, 30-100 kDa and above 100 kDa.

isoelectric focusing

The shark cartilage preparation (46 mL of 1 kg / L filtrate) was dialyzed overnight at 4 ° C with 4 L of 5% glycerol in clear water using a # 7 MWCO 3500 KDa pore Spectra membrane (Spectrum 132110). The dialyzed solution was treated with 2.75 mL of pH 3.5-10.0 Ampholite solution (Pharmacia # 80-1125-87) and 0.5 g of 3 - [(colamidopropyl) dimethylammonio] -1-propanesulfonate (Sigma C3023). stirred. The volume was made up to 55 ml with clean water. The solution was placed in a rotor. The isoelectric focusing was performed at 4 ° C, 12 Watt constant power (3000xi Biorad

165-0554), constant temperature was maintained by continuous circulation of water. At the beginning of the separation, the voltage was 380 and the current was 31 amps. After the current stabilized (14 mAen), the voltage increased to 870 volts. At this point, the isoelectric focusing was stopped and 20 fractions were collected.

faction Volume (ml) pH 1 3.7 3.56 2 2.1 4.01 3 2.2 4.18 4 2.3 4.31 5 2.2 4.63 6 2.1 5.03 7 2.5 5.30 8 2.1 5.50 9 2.4 5.81 10 2.5 6.26 11 2.3 7.00 12 2.4 7.29 13 2.4 7.64 14 2.5 7.94 15 2.3 8.32 16 2.5 8.62 17 2.4 8.94 18 2.9 9.30 19 3.1 9.88 20 3.6 10.71

Proteins were identified on the basis of their molecular weights determined by electrophoresis gel (Laemmli, U.K. (1970) Natúré (Lond.) 227; 680).

Fractions were diluted four-fold with buffer (see Laemmli) and aliquots of 8 µl were withdrawn and assayed by electrophoresis under non-reducing conditions. The electrophoresis profile of the pre-isoelectric focusing material and fractions is shown in Figure 8.

The fractions were filtered through a Millipack-60 sterile filter with a porosity of 0.22 µm in a laminar flow cabinet and bottled under sterile conditions.

The protein content of the fractions was determined by the Lowry dose procedure. The effect of 1 kg / 2 l solutions (crude cartilage mass per liter filtrate) on ZR75-1 cells was examined in different concentrations of cell culture medium.

Summary of results

Test 1:

The filtrate was lyophilized and analyzed by FPLC. Hypoplastic activity could not be measured (no data).

HU 220 784 Β1

Test 2:

Investigation of Rotophor Fractions: Protein Determination

identified fractions Isoelectric point Middle- value molecular Weight (KDa) 7-8-9-10. 5.30 to 6.26 5.78 29 ± 1 7-8-9. 5.30 to 6.26 5.68 60 ± l 12-13-14. 7.29 to 7.94 7.62 48 ± 1 13-14. 7.64 to 7.94 7.79 35 ± 1

Test 3:

Examination of FPLC fractions:

factions molecular Weight 6 and 7 1-2.5 kDa

Test 4:

Examination of ΙΟΟμΙ fractions separated by Amincon molecular sieve:

Because concentration molecular Weight (KDa) Inhibition on ZR75-1 cell cultures 100 pg / ml MS> 100 64% 100 pg / ml 30 <MS <100 114% 100 pg / ml 10 <MS <30 127% 400 pg / ml MsCl 149%

Fractions 6 and 7 of FPLC contained very low molecular weight active components: 1 to 2.5 kDa.

The hypoplastic effect of the fractions can be up to 33,000 times that of the lyophilisate. The above results show that lyophilization completely destroyed and / or inhibited the activity of the proteins in the eluate, whereas no abolition occurred when the solid extract was lyophilized.

Further identification of the active components of the eluate:

Effective fractions obtained from the following molecular weight range (tested on ZR75-1 cells) were determined from the same filtrate (1 kg / l) by another type of purification procedure on a Superose-12 column 10 mm in diameter and 30 cm long, as described above. flow rate was set at 1 ml / min. 45 fractions of 1 ml were collected.

20-21. Fractions: The activity of the fractions corresponded to a molecular weight of 70-120 kDa

Fraction 22: The activity of the fractions corresponded to a molecular weight of 60-70 kDa

29-32. Fractions: The activity of the overlapped fractions corresponded to a molecular weight of 35-46 kDa

34-35. Fractions: The activity of the fractions corresponded to a molecular weight of 29 kDa

38-39. Fractions: The activity of the fractions corresponded to a molecular weight of 1-2.5 kDa.

Examination of specificity

To investigate the specific effect on tumor cells, the filtrate was ultrafiltered and then tested on other parent cell derived cells, namely human TENON connective tissue cells (HTF), which are regular connective tissue cells.

B. In vitro

the. Patients

Only HTF from two patients (one neovascular glaucoma, NVG, and one primary open-angle glaucoma POAG) was used.

b. Secondary breeding and maintenance of HTFs

Both confluent cultures were washed and separated with 0.5 ml of 0.05% trypsin / 0.5 mM EDTA (Gibco 610-5300 hp) for 5-10 minutes at 37 ° C. 1.5 ml DME / F-12 medium containing 15% FBS was added to neutralize trypsin / EDTA.

Cell association was accomplished by crushing and transferring to a 25 cm ² T-flask supplemented with supplemental medium containing 10% FBS. After the culture reached confluence, the HTF was placed in a 75 cm ² and finally a 180 cm ² T flask. When sufficient cells were generated, some of these were used for the experiments described above, and the rest were frozen to retain the same cells for further experiments.

c. Experimental specifications

After confluence, cells from one patient were cultured in two or three identical 180 cm ² T-flasks and then split as described above. After briefly centrifugation at low speed, cells were counted in a ZMI Coulter 216013 with 256-Channelelyzer.

For the following in vivo experiments, approximately 50,000 cells were inoculated into 1 ml DME / F12 medium containing 1% FBS in each 16 mm plate and 12-well plate. Seventeen hours after inoculation, 1 ml of fresh medium containing 1% FBS ("absolute" control) was added identically. Depending on the design of the experiment (see above and below) GFet (growth factor) or 1 kg / 21 (cartilage weight / volume of water) filtrate solution was added to 1% FBS medium and filtered under sterile conditions. On this day (Day 0), a number of cell samples were counted to determine the efficiency of plating (equal to or greater than 95%). After forty-eight hours from the start of the experiment, the cells were rinsed, distributed as described above, and counted again. The number of cells is expressed as a percentage of the values determined for the "absolute" control.

Each "absolute" or positive control group containing 1% or 5% FBS and each experimental group containing 1% FBS and a given amount of GF or cartilage filtrate consisted of three to three samples.

The experiments were performed on cells of one or both patients and were repeated at least twice.

The effect of growth factors (Gf) and cartilage filtrate on fibroblast proliferation was compared with that of 5% FBS.

HU 220 784 Β1

In the experiments, 10-100 ng / ml GF, porcine pDGF (pPDGF) and recombinant human bFGF (hr bFGF) (gift from Farmitalia Carlo Erba (Milan, Italy) to Dr. P. Brazeau) were added to 1% FBS. Forty-eight hours after the start of the experiment, cells were lysed with Trypsin-EDTA and counted on a Coulter counter. The value triples (columns 1, 2 and 3) in the table below are one-twentieth of the values determined at each well

Patient: Glaucoma Gender: Male Age: 53 HTF —1. day: Number of cells per well: 46170 DME / F12 - 1% FBS-1% Pen. Strep

Day 0: Number of cells per well: 65214

DME / F12 - 1% FBS-1% Pen. Strep

Day 1: Number of cells per well: 62548

DME / F12 - 1% FBS-1% Pen. Strep

equal. 10 2 days: Number of cells per well: Sample / plate First Second Third ST. SEM % control growth Tray # 1 DME / F12 FBS 1 Day 0 1% FBS 3.019 2,862 2.853 65.214 71 2 + Day 1 1% FBS 2,711 2,973 2.693 62.548 1,655 3 +2. Sun 1% FBS 2,284 2,400 2,191 51.333 1,655 100 4 +2. Sun 3.084 2,834 3,115 67.446 1,627 131 5% FBS ♦♦ Tray # 2 DME / 12 PDGF 5 control 2.558 2,181 2,216 51.931 2,199 100 (1% FBS) 6 1 ng / ml 1% FBS 2,425 2.580 56.056 1,228 108 7 10 ng / ml 4.080 3.975 4.282 92.116 1,648 177 1% FBS 8 100 Ng / ml 4,625 4.356 4.450 100.285 1,442 193 1% FBS ♦ ** Tray # 3 DME / F12 b-FGF 9 control 2.915 2,533 2.502 59.360 2,429 100 (1% FBS) 10 1 ng / ml 1% FBS 2,744 2.554 2.761 60.174 1,213 101 11 10 ng / ml 3.606 3,143 3.193 74.234 2,683 125 1% FBS 12 100 ng / ml 4,064 3.033 3.026 75.585 6.307 127 1% FBS Tray # 4 DME / F12 PORC 13 control 2,826 2.566 2,486 58.822 1.877 100 (1 kg / 21) (1% FBS) (filtered) 14 1 μΐ / ml 1% FBS 2,729 2,576 2.575 58.837 936 100 15 10 μΐ / ml 1% FBS 2.643 2,493 2,584 57.643 798 98 16 100 μΐ / ml 2,918 2.883 2.766 58.483 2,461 99

1% FBS ** P <0.02 *** P <0.01 as determined by Student-Fisher test

HU 220 784 Bl

Growth factors, such as PDGF (platelet-derived growth factor) and bFGF (basic fibroblast growth factor), had a stimulatory effect on HTF, whereas no positive or negative effect was observed in cells cultured in the presence of cartilage filtrate (1 kg / 2 L). No hypoplastic effect was observed. This indicates that the hypoplastic effect of the filtrate is tumor cell specific and has no measurable effect on normal cells. The same cartilage extract does not affect HSF (human skin fibroblast cells; no data) for other types of fibroblast cells. Although not tested, the lyophilisate is not expected to have an effect on normal cells.

Clinical trials

Prior to conducting the preliminary clinical studies, the crude filtrate obtained by ultrafiltration was concentrated 2 to 20 times to obtain a concentrated active filtrate. The concentrates were prepared on a 1000 Da porous tangential flow filtration column which reduced the filtrate volume to half or one twentieth. The concentrated filtrate was filtered through a 0.22 µm porosity filter. When the cartilage was subjected to the alternative centrifugation method (CEPA centrifuge with 30 M porous membrane), the concentrated extract was obtained at a concentration of ten times that of the protein was almost the same as that of the twenty-fold concentrated extract above (14 mg / ml). / ml (laboratory scale procedure). The sterile 10x concentrated filtrate was dispensed into sterile vials into aliquots of 7 ml (about 85 mg white), stored at -80 ° C overnight and stored at -20 ° C until use. The main difference between the crude and concentrated filtrates was their protein content. Note that the method used to determine the protein content is to measure all nitrogen compounds and not just the protein (Kjeldahl method). This explains why the protein concentration does not increase proportionally to the volume concentration, as opposed to the determination of protein content by the Lowry method. It is expected that low molecular weight nitrogen compounds will be eliminated along with water in the concentration step.

Concentrated filtrate was used to treat vascular dependent diseases. We investigated two different types of vascular-dependent diseases in human medicine; the first type is dermatological disorders (psoriasis), the second type is cancer (prostate cancer).

From dermatological diseases, psoriasis was chosen. Among psoriatic cases, cases of both complicated and uncomplicated hyperkeratosis were investigated. Concentrated cartilage filtrate did not substantially affect the keratosis component of the disease, the drug mixture targeted the vascular component. The following examples illustrate this statement:

A patient with prostate cancer volunteered to attempt a 10-fold concentrated cartilage filtration. This patient underwent a series of successful conventional therapies, but their success was temporary. The patient started consuming the cartilage extract shortly after relapsing into the disease.

The results presented below are very encouraging, and the applicability of the crude filtrate and its fractions appears to be promising not only for the treatment of the specific diseases studied, but for all diseases dependent on vascularization. Against vascular disease, the cartilage extract of the present invention is expected to be effective when the composition administered contains an effective amount thereof and is in a form suitable for administration. Therefore, it is to be understood that the present invention is not limited to the following individual formulations, as will be apparent to those skilled in the art from a variety of formulations, depending upon the proper route of administration and targeting the affected tissue. The compositions may be administered by a variety of routes, e.g., topically, orally, sublingually, rectally, intravenously, intramuscularly, by diffusion, and the like.

psoriasis

We have prepared a dermatological formulation of the following composition and attempted to demonstrate efficacy in patients with psoriasis:

- Emulgade ™ CLB 29% by weight

- 20X crude filtrate 69.5% by weight

Germaben ™ II 1% by weight and

- Lavandula Augustofolia 0.5% by weight.

Emulgade ™ CLB, a mixture of stearate esters, fatty alcohols and nonionic emulsifiers (available from Henkel Canada Ltd.) were heated to 65-70 ° C with stirring. After the heating was stopped, stirring was continued.

When the mixture was cooled to 45 ° C, Lavandula Augustifolia Essential Oil and Germaben ™ II Preservative (Diazonidyl Urea 30%, Methyl Paraben 11%, Propyl Paraben 3% and Propylene Glycol 56%; Sutton Laboratories, NJ, USA) were added. added. When the temperature of the mixture dropped to 30 ° C, the cartilage extract was added. The resulting composition is a uniform, non-greasy cream. By varying the Emulgade ™ content, other forms of dermatological compositions of different viscosities can be prepared according to the manufacturer's instructions (milk, lotion, ointment). If another carrier or excipient is used, a paste, gel or other form of transdermal preparation can be prepared.

The above formulation was given twice daily for 12 weeks to a group of 10 patients (topical application), who were suffering from psoriasis and, although responding to previously tried conventional therapies, became difficult to heal after a while. Patients with symmetrically sympathetic psoriasis on both sides were selected for the study. Duplicate experiments were performed, neither the dermatologist nor the patient knew which side to treat with the cartilage extract formulation and which with the control formulation. Significant improvement was seen in 5 patients without psoriasis not complicated by hyperkeratosis. Photographs of body parts of two patients are shown in Figure 9A. 9B and 9B. FIGS. 9A. Fig. 6A shows a patient with hyperkeratosis psoriasis with a significant decrease in non-pruritic erythema after one month of treatment.

HU 220 784 Β1 lubrication, but hyperkeratosis remained significant. The second patient, whose psoriasis was not complicated by hyperkeratosis (Fig. 9B), showed a significantly greater improvement after 3 months of treatment. Although psoriasis is a multifactorial disease, it is believed that the patient's response depends on the importance of the factor for the development and maintenance of his condition. It is likely that better results could be achieved if the product were supplemented with therapeutic agents that would target other factors involved in the disease (keratolytic agents, anti-inflammatory agents, antihistamines, immunosuppressants, etc.).

For example, the composition may be modified by adding an effective amount of a keratolytic agent. The additional therapeutic agent may be administered separately, simultaneously or alternately with the topical formulation, and the additional treatment may not be similar.

The above formulation showed no systemic effect (the effect was limited to the treated part) and no secondary (secondary) effects were observed despite the high proportion of cartilage extract.

acne

Despite the authors' knowledge that acne is not classified as a disease or disorder dependent on the vascular component, attempts have been made to treat patients suffering from such disease with a liquid cartilage extract. In patients with acne, the cartilage extract was tested with the following gel composition:

Carbopol ™ 1.2% Cleaned water 77.2% NaOH 0.3% Phenoxetol ™ 0.3% Tween 80 ™ 0.3% 2 χ porcextraktum 20.0% 40 χ aloe extract 0.5%. A 2 χ porctax extract 9- -12 mg / ml protein content-

out. This formulation has led to a remarkable improvement in the condition of patients with more or less severe forms of acne (inflammatory acne and cystic acne); data not shown.

These surprising results are caused either by the anti-angiogenic effect (which refers to the role of the germ component in acne) or by the active ingredient having a non-anti-angiogenic action. It is recalled that at least one other effect of the same extract, besides inhibition of vascularization, is known: it has a direct hypoplastic effect on cancer cell lines.

Cancer

A patient with prostate cancer was treated with a 10-fold filtrate. The patient was diagnosed with adenocarcinoma in 1986. Radiotherapy was used at this time. In 1991, the patient had a PSA (Prostate Serum Antigen) level of 138 pg / L, whereas the highest acceptable normal limit was 4 pg / L. Thereafter, the patient underwent a completely different therapy, castration combined with anti-androgen therapy (Euflex ™). This treatment was effective for 3 years, after which time PSA levels increased again. Since June 1994, the patient has been consuming filtrate at a concentration of ten times daily (sublingually, about 75 mg protein / 7 ml distilled water, equivalent to about 1-1.5 mg / kg body weight / day). Based on the results obtained with DMBA treated animals (see above), a significant portion of this dose is likely to be ingested and absorbed in the gastrointestinal tract. PSA levels were reduced from 12 to 0.9 pg / ml (final results were determined in December 1994). The dosage may be varied according to the route of administration, the bioavailability of the active ingredient, and the potency required to control the pathology. At this time, the substance was shown to be non-toxic in rats (see sample above) and humans (data not shown).

In other in vivo experiments in DMBA-treated rats, the dose ratio of liquid extract was about 190-220 mg / kg protein to body weight, presumably contributing significantly to the reduction in blood vessels in cancerous areas (55% when combined with a much higher dose of lyophilisate). Therefore, it is anticipated that a mean range (ED ₅₀ ) per kg of body weight per day for the reduction or elimination of vasculature in the treatment of cancer will be about 1 to about 200 mg per day.

The results show that liquid cartilage extract has great potential in the treatment of vascular-dependent diseases. The amount and mode of administration of the cartilage extract may be varied to meet specific needs. It is believed that the crude filtrate containing the active ingredient is also effective. Further characterization of these fractions has not yet occurred.

It should be noted that the cartilage extract can be applied to the skin in a topical formulation over a wide dosage range based on protein content. For example, in the two special groups of cases studied, the ratio of final protein concentration in the preparation to 4-5 and the filtrate dilution ratio to 35 in each case of psoriasis and acne is expected to be as low as dermatological and anticancer agents. the minimum protein concentration in the crude filtrate may be very low (less than 0.1 mg / ml). The lower dose range depends on the availability, the penetration of the active agent into the area to be treated, the efficiency of the uptake of the active ingredient, the response of the tissues, or how sensitive they are to anticoagulants. The upper limit of the protein concentration in formulations used for various purposes is not yet known. The highest concentration we used was about 9 mg / ml protein for psoriasis and about 12 mg / ml for prostate cancer in the 7 ml dose unit. Since it cannot be lyophilized without losing the activity of the crude filtrate, it is believed that the upper dose range is determined by the concentration at which the crude filtrate can still be produced, i.e., the concentration of the syrup still obtainable.

HU 220 784 Bl

Necessary materials:

- refrigerators

- surgical equipment

- meat grinder

plastic bags 5

- industrial mixer (purchased from Fischer Scientific, Waring 3 stage mixer)

- water purification system (inverse osmosis and 0.1 pm filtration; Continental Water System 91089, Model PRE 2202, Modulab Bioscience 10 RQ / Polishing System, Fischer scientific, Montreal Quebec). This system provides high quality apyrogenic water.

- Mettler Precision Balance, Series AE, Fischer

Scientific 15

- RC-285 Sorvall Centrifuge, DuPont (Canada)

- CEPA centrifuge

- Nylon bag with a porosity of 30 pmol

- autoclave (Sanyo automatic vaporizer,

MAC 35OP model) 20

- 500 ml Nalgene container sterilized at 132 ° C for 10 minutes and dried for 35 minutes

- Whatman Reeve Angel conical filter, porosity 24 pm

ultrafiltration column (molecular weight range 500 kDa 25 (and 1 kDa if applicable); surface 25 square feet; flow 130 rpm; inlet pressure 30 psi; outlet pressure 5 psi; Koch Membrane System, Wilmington, MA, USA)

- Health Centrifuge Pump (Monarch Injection ACE-S100 Model A) to provide a flow rate of 130 rpm

- sterile cabin (NuAire laminar flow cabin, Ingram & Bell)

Millipack-60 0.22 pm sterile filter 35

- sterile, clear or amber bottles

- DC-10 Amicon concentrator

- Rotofor Biorad 170-2950

- Amicon filters SIOY10, SIOY30 and SIOY100

With limits of 10, 30 and 100 kDa 40

- FPLC Pharmacia 216007 (Pharmacia 216014 Computer)

- Hilstand S-300 26mm / 60cm (Pharmacia)

- Superose S-12 10mm / 30cm (Pharmacia)

- Labconco 10273 The lyophilizer. 45

The present invention has been described above and it is to be understood that no modifications or substitutions of any aspect of the invention to the person skilled in the art, or the use of equivalents, are apparent to those skilled in the art. These obvious variants also form part of the invention.

Claims (27)

A liquid extract comprising a fraction of a supernatant of a substantially complete shark cartilage aqueous homogenate produced in a centrifugation step and a subsequent fractionation step on a membrane having a nominal molecular weight cutoff of about 500 kDa, wherein the fraction produced has a molecular weight of less than 500 Fraction II contains a component with direct antitumor activity. The liquid extract according to claim 1, comprising essentially a fraction of a supernatant of the whole shark cartilage aqueous homogenate produced in the centrifugation step and subsequent fractionation on membranes with nominal molecular weights of 1 kDa and 500 kDa, which fraction is between 1 kDa and 500 kDa. molecule-rich and which fraction contains a direct antitumor component. The liquid extract of claim 1, which has the following composition: fats protein moisture sodium potassium calcium magnesium zinc and iron 0.10% 1.77% 98.8% 33.6 mg / 100 g 39.2 mg / 100 g 2.0 mg / 100 g 1.1 mg / 100 g traces. v) The liquid extract of claim 1, further comprising: (i) a liquid fraction having a molecular weight range of 1 kDa to 2.5 kDa as determined by Rapid Protein Liquid Chromatography (FPLC); ii) a liquid fraction having a molecular weight of about 29 kDa after electrophoresis after rotor separation, with an isoelectric point between 5.3 and 6.26; iii) a liquid fraction having a molecular weight of about 35 kDa after electrophoresis after rotor separation, with an isoelectric point between 7.64 and 7.94; iv) a liquid fraction having a molecular weight of about 48 kDa after electrophoresis after rotor separation, with an isoelectric point between 7.29 and 7.94; The liquid fraction, which after electrophoresis has a molecular weight of about 60 kDa after rotor separation, has an isoelectric point between 5.30 and 6.26. The liquid fraction of the liquid extract of claim 1, wherein (i) a molecular weight of between 1 kDa and 2.5 kDa as determined by FPLC; or (ii) a molecular weight of about 29 kDa after electrophoresis after rotophor separation, a molecular weight of about 35 kDa after electrophoresis after separation of rotophores; has an isoelectric point of 7.64 to 7.94 or (iv) has a molecular weight of about 48 kDa after electrophoresis after rotor separation, and an isoelectric point of 7.29 to 7.94; or v) having a molecular weight determined by electrophoresis after rotophor separation, of about 60 kDa, an insulating point of between 5.30 and 6.26. HU 220 784 BI The liquid extract of claim 1, further comprising: (i) a liquid fraction which, after separation by rotophores Has a molecular weight determined by FPLC of between 70 kDa and 170 kDa, ii) a liquid fraction which, after separation by rotor Molecular weight determined by FPLC between 60 kDa and 70 kDa, iii) liquid fraction, which after rotophor separation Molecular weight determined by FPLC between 35 kDa and 46 kDa, iv) liquid fraction after Its molecular weight as determined by FPLC is about 29 kDa, (v) a liquid fraction which, after separation by rotophores It has a molecular weight of 1 kD and is determined by FPLC 2.5 kDa. The liquid fraction of the liquid extract of claim 1, wherein (i) a molecular weight range of 70 kDa and 120 kDa determined by FPLC after rotophoric separation; or (ii) a molecular weight range of between 60 kDa and 46 kDa after rapid separation of rotophores by liquid chromatography of 60 kDa and 70 kDa; or (iv) having a molecular weight of approximately 29 kDa as determined by rapid protein chromatography after rotor separation; or (v) after separation by rotor phosphor the molecular weight of its rapid protein determined by liquid chromatography is between 1 kDa and 2.5 kDa. 8. A fluid fraction according to any one of claims 1 to 4, which comprises a component having a direct antitumor activity against hormone dependent breast cancer cells in vitro. 9. A process for the preparation of a shark cartilage liquid extract comprising the steps of: i) homogenizing the shark cartilage in a non-denaturing aqueous solution to form a mixture of shark cartilage particles and crude liquid extract, wherein the particles are 500 µm or less in size, ii) separating the crude liquid extract from the particles and iii) separating the crude liquid extract A final liquid extract containing cartilage molecules having a molecular weight of kDa or less is isolated. The process of claim 9 wherein the non-denaturing solution is an aqueous solution and the steps are performed at about 4 ° C. 11. The process of claim 9 or 10, wherein step i) is performed for about 30 minutes. 12. A 9-11. A process according to any one of claims 1 to 3, wherein the shark cartilage and the non-denaturing aqueous solution are used in a ratio of about 1 kg wet cartilage weight to 1 to 21 solutions. 13. A 9-12. The process according to any one of claims 1 to 4, wherein step ii) is performed by centrifugation. 14. A 9-12. A process according to any one of claims 1 to 4, wherein a further step iv) is carried out in which the final liquid extract is concentrated to produce a concentrated liquid extract containing cartilage molecules having a molecular weight of between 1 kDa and 500 kDa. 15. A 9-13. A process according to any one of claims 1 to 4, wherein a further step (iv) is carried out in which the final liquid extract is fractionated to obtain a liquid fraction of the liquid extract. 16. The process of claim 15, wherein the fractionation step is carried out by FPLC to obtain a liquid fraction containing cartilage molecules having a molecular weight of between 1 kDa and 2.5 kDa. 17. The process of claim 15 wherein the fractionation step is performed by rotor separation and electrophoresis to provide a liquid fraction in which the cartilage molecules a molecular weight of about 29 kDa and an isoelectric point of 5.3 to 6.26, or has a molecular weight of about 35 kDa and an isoelectric point between 7.64 and 7.94, or has a molecular weight of about 48 kDa and an isoelectric point between 7.29 and 7.94 or has a molecular weight of about 60 kDa and an isoelectric point of between 5.30 and 6.26. 18. The process of claim 15 wherein the fractionation step is performed by rotor separation and FPLC to provide a liquid fraction in which the cartilage molecules - molecular weight between 70 kDa and 120 kDa, or - molecular weight between 60 kDa and 70 kDa, or - molecular weight between 35 kDa and 46 kDa, or a molecular weight of about 29 kD, or - molecular weight between 1 kDa and 2.5 kDa. 19. The porcine extract, which is illustrated in FIGS. A process according to any one of claims 1 to 6. 20. A shark dust extract containing at least one biologically active component having a molecular weight of less than 500 kDa, exhibiting antiangiogenic activity and in vitro antitumor activity, the latter activity being determined by an antitumor activity assay in which angiogenesis is not involved in cell proliferation, and in vivo exhibits antitumor activity in tumors where angiogenesis plays a role in cell proliferation; which in vivo anti-tumor effect may be independent of the anti-angiogenic effect. The shark powder extract of claim 20, comprising at least one biologically active component having a molecular weight of from 1 kDa to 500 kDa. 22. The shark powder extract of claim 20, comprising at least one biologically active component comprising at least one biologically active component having HU 220 784 Bl - has a molecular weight in the range of 1 kDa to 2.5 kDa, - has a molecular weight in the range of 29 kDa, - has a molecular weight in the range of 35 kDa, - has a molecular weight in the range of 35 kDa to 46 kDa, - has a molecular weight in the range of 48 kDa, - has a molecular weight in the range of 60 kDa, has a molecular weight in the range of 60 kDa to 70 kDa, and / or has a molecular weight in the range of 70 kDa to 120 kDa. The shark powder extract of claim 20, wherein the at least one biologically active component comprises at least one biologically active component having - an isoelectric point between 5.3 and 6.26, an isoelectric point between 7.64 and 7.94, and / or - an isoelectric point between 7.29 and 7.94. A pharmaceutical composition for the treatment of dermatological disorders, acne, cancer or diseases characterized by one or more etiological components, namely, tumor growth or vascularization, which are characterized in that the compounds of any of claims 1-3, 19 or 20-23. An effective amount of a cartilage extract according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. 25. A process for the preparation of a liquid shark powder extract, comprising: a) homogenizing the shark cartilage particles in an aqueous solution having a non-denaturing effect on the biologically active components to form solid shark cartilage particles having a size of 500 µm or less; b) extracting the bioactive components in this aqueous solution to form a mixture of solid particles and crude liquid cartilage; c) separating the crude liquid extract from the solid particles; d) fractionating the crude liquid extract to form a crude liquid extract subfraction containing molecules having a molecular weight of less than 500 kDa; and e) further fractionating the crude liquid extract subfraction on a membrane with a nominal molecular weight cutoff of 1 kDa to form a fraction containing molecules of less than 1 kDa. A cartilage fraction comprising molecules having a molecular weight of less than 1 kDa produced by the process of claim 25. A composition comprising a cartilage action according to claim 26 and a pharmaceutically acceptable carrier and / or excipient.